The invention relates to novel PEGylated compounds and a process for formation of PEGylated compounds. More specifically, the present invention is related to novel PEGylated compounds, primarily PEGylated amino acid monomers and their use in the formation of advanced peptides. The invention provides synthesized amino acids of glutamine and lysine that are directly PEGylated with multiple small, monodisperse PEGs. These amino acids are readily incorporated into peptides for a range of different applications.
PEGylation is a process of attaching strands of polymeric polyethylene glycol (PEG) to molecules, most typically peptides, proteins, and antibody fragments, that can help to meet the challenges of improving the safety and efficiency of many therapeutics. PEGylation produces alterations in the physiochemical properties including changes in conformation, electrostatic binding, hydrophobicity, and the like. These physical and chemical changes increase systemic retention of the therapeutic agent. PEGylation can also influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.
By increasing the molecular weight of a molecule, PEGylation can impart several significant pharmacological advantages over the unmodified form. Advantages include improved drug solubility, reduced dosage frequency without diminished efficacy, potentially a reduced toxicity, an extended circulating life, increased drug stability and enhanced protection from proteolytic degradation.
PEG is a particularly attractive polymer for conjugation. The specific characteristics of PEG moieties relevant to pharmaceutical applications are water solubility, high mobility in solution, lack of toxicity and low immunogenicity, ready clearance from the body and altered distribution in the body.
The overall PEGylation processes used to date for protein conjugation can be broadly classified into two types, namely a solution phase batch process and an on-column fed-batch process. The simple and commonly adopted batch process involves the mixing of reagents together in a suitable buffer solution, preferably at a temperature between 4° C. and 6° C., followed by the separation and purification of the desired product using a suitable technique based on its physicochemical properties, including size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and membranes or aqueous two phase systems.
The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates and carbonates. In the second generation PEGylation chemistry more efficient functional groups are made available for conjugation such as aldehyde, esters, amides etc.
As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in the need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and N-hydroxysuccinimide (NHS) esters.
Third generation PEGylation agents, where the shape of the polymer has been branched, Y shaped or comb shaped are available which show reduced viscosity and lack of organ accumulation.
Dozens of peptide drugs are on the market but the difficulty in forming PEGylated peptides is evident by the lack of any appreciable PEGylated peptides and currently there are no PEGylated peptides of commercial significance. Like with larger proteins, PEGylation of peptides can increase solubility and reduce cleavage of the peptides by proteases in the blood, both of which are severe problems for peptide drugs. For example, Victoza®, (Iiraglutide, Novo Nordisk) the first non-insulin peptide blockbuster drug, is a GLP-1 mimic; the half-life of GLP-1 in blood is 90 seconds.
Most PEGylation strategies are designed for large proteins wherein long PEGs are used to impart the desired properties. The long PEGs, however, are too big for peptides. In practice, peptide modification sites are limited primarily to lysines and the N-terminus, although all the same reactive side chains of proteins (cysteines, aspartic acids, glutamic acids, serines, threonines, histidines) could in theory be modified.
Peptides are made in the lab using Solid Phase Peptide Synthesis (SPPS), an extremely straightforward technique that has been in use since the 1960s. Since peptides, like proteins, are PEGylated after the whole peptide has been synthesized, site-specifically PEGylating peptides requires “orthogonal protection” synthetic schemes that typically require Ph.D. level scientists to design, execute and employ more costly and dangerous reagents. The difficulty and expense has limited the ability of those of skill in the art to even study the potential for PEGylated peptides with any degree of specificity thereby hindering even the exploration of the potential impact of these materials on the market.
As discussed above, amino acids and peptides play important roles as drug compounds, in nutrition, as well as in biotechnology and other applications. Peptides, however, suffer from poor solubility, protease degradation in the body, and short shelf-lives. These problems have been very successfully solved for large proteins primarily in the pharmaceutical industry via PEGylation with large PEG polymers. The large PEG polymers, however, are too large and polydisperse for smaller peptides, thus leaving those of skill in the art without an adequate way to provide tailored PEGylated peptides for study and exploitation.
Moreover, prior art PEGylation strategies involve PEGylating amino acids, sometimes using long, polydisperse PEGs during peptide synthesis using elaborate chemical strategies that often greatly alter the original properties of the amino acids. The limited size of peptides renders these techniques unsuitable. It is difficult to control PEGylation location on the peptide and virtually impossible to form di- and tri-PEGylated amino acids in a peptide.
PEGylated lysine has been described in the past wherein the PEGylated lysine has a single PEG chain. Unfortunately, to achieve adequate functionality the PEG group length is often sufficiently long to wrap around the peptide thereby inhibiting other reactions. Furthermore, PEGylation is typically done using amide-bond-forming chemistry which inhibits the ability of the lysine to function as a salt bridge thereby mitigating a secondary function of the lysine functional group.
The present invention provides novel amino acids, novel peptides incorporating the amino acids, and a method of preparing novel peptides wherein PEGylation can be controlled, both in terms of the location on the peptide chain and in terms of the density of PEG groups, neither of which were easily controlled previously.